Identification of an asymmetrically localized sensor histidine kinase responsible for temporally and spatially regulated transcription.

Caulobacter crescentus undergoes asymmetric cell division, resulting in a stalked cell and a motile swarmer cell. The genes encoding external components of the flagellum are expressed in the swarmer compartment of the predivisional cell through the localized activation of the transcription factor FlbD. The mechanisms responsible for the temporal and spatial activation of FlbD were determined through identification of FlbE, a histidine kinase required for FlbD activity. FlbE is asymmetrically distributed in the predivisional cell. It is located at the pole of the stalked compartment and at the site of cell division in the swarmer compartment. These findings suggest that FlbE and FlbD are activated in response to a morphological change in the cell resulting from cell division events.

Positional information during Caulobacter cell differentiation.

The formation of two distinct daughter cells upon division of the bacterium Caulobacter crescentus is the result of asymmetry in the predivisional cell, in part due to localization of both flagellar and chemotaxis proteins to the swarmer cell pole. Recent evidence suggests that both localized transcription and protein targeting directed by specific amino acid sequence are involved in the localization.

Relationship between myocardial metabolites and contractile abnormalities during graded regional ischemia. Phosphorus-31 nuclear magnetic resonance studies of porcine myocardium in vivo.

The mechanisms responsible for changes in myocardial contractility during regional ischemia are unknown. Since changes in high-energy phosphates during ischemia are sensitive to reductions in myocardial blood flow, it was hypothesized that myocardial function under steady-state conditions of graded regional ischemia is closely related to changes in myocardial high-energy phosphates. Therefore, phosphorus-31 nuclear magnetic resonance spectroscopy was employed in an in vivo porcine model of graded coronary stenosis. Simultaneous measurements of regional subendocardial blood flow, high-energy phosphates, pH, and myocardial segment shortening were made during various degrees of regional ischemia in which subendocardial blood flow was reduced by 16-94%. During mild reductions in myocardial blood flow (subendocardial blood flow = 83% of nonischemic myocardium), only the ratio of phosphocreatine to inorganic phosphate (PCr/Pi), Pi, and [H+] were significantly changed from control. PCr, ATP, and PCr/ATP were not significantly reduced from control with mild reductions in blood flow. Changes in myocardial segment shortening were most closely associated with changes in PCr/Pi (r = 0.94). Pi and [H+] were negatively correlated with segment shortening (r = -0.64 and -0.58, respectively) and increased over twofold when blood flow was reduced by 62%. Thus, these data demonstrate that PCr/Pi is sensitive to reductions in myocardial blood flow and closely correlates with changes in myocardial function. These data are also consistent with a role for Pi or H+ as inhibitors of myocardial contractility during ischemia.

A developmentally regulated Caulobacter flagellar promoter is activated by 3' enhancer and IHF binding elements.

The transcription of a group of flagellar genes is temporally and spatially regulated during the Caulobacter crescentus cell cycle. These genes all share the same 5' cis-regulatory elements: a sigma 54 promoter, a binding site for integration host factor (IHF), and an enhancer sequence, known as the ftr element. We have partially purified the ftr-binding proteins, and we show that they require the same enhancer sequences for binding as are required for transcriptional activation. We have also partially purified the Caulobacter homolog of IHF and demonstrate that it can facilitate in vitro integrase-mediated lambda recombination. Using site-directed mutagenesis, we provide the first demonstration that natural enhancer sequences and IHF binding elements that reside 3' to the sigma 54 promoter of a bacterial gene, flaNQ, are required for transcription of the operon, in vivo. The IHF protein and the ftr-binding protein is primarily restricted to the predivisional cell, the cell type in which these promoters are transcribed. flaNQ promoter expression is localized to the swarmer pole of the predivisional cell, as are other flagellar promoters that possess these regulatory sequences 5' to the start site. The requirement for an IHF binding site and an ftr-enhancer element in spatially transcribed flagellar promoters indicates that a common mechanism may be responsible for both temporal and polar transcription.

Cell cycle control of cell morphogenesis in Caulobacter.

In Caulobacter crescentus, morphogenic events, such as cytokinesis, the establishment of asymmetry and the biogenesis of polar structures, are precisely regulated during the cell cycle by internal cues, such as cell division and the initiation of DNA replication. Recent studies have revealed that the converse is also true. That is, differentiation events impose regulatory controls on other differentiation events, as well as on progression of the cell cycle. Thus, there are pathways that sense the assembly of structures or the localization of complexes and then transduce this information to subsequent biogenesis or cell cycle events. In this review, we examine the interplay between flagellar assembly and the C. crescentus cell cycle.

Protein localization during the Caulobacter crescentus cell cycle.

New research on bacterial cells has demonstrated that they have a dynamic and complex subcellular organization. Work in Caulobacter crescentus shows that essential and nonessential proteins localize to discrete positions in the cell as a function of cell-cycle progression. The flagellum and chemotaxis receptor are asymmetrically localized to a single pole in the predivisional cell by coordinated proteolysis and transcriptional regulation. Cell type- and compartment-specific localization of the CtrA global transcriptional regulator is essential for proper cell-cycle progression, and subcellular localization of key chromosome partitioning proteins is correlated with proper nucleoid segregation. Given this structural complexity, we are driven to ask how localization is achieved, and to what end.

Nuclear magnetic resonance imaging-guided phosphorus-31 spectroscopy of the human heart.

Phosphorus-31 nuclear magnetic resonance spectroscopy can determine the status of high energy phosphates in vivo. However, its application to human cardiac studies requires precise spatial localization without significant contamination from other tissues. Using image-selected in-vivo spectroscopy (ISIS), a technique that allows three-dimensional localization of the volume of interest, 12 subjects were studied to determine the feasibility and reproducibility of phosphorus-31 spectroscopy of the human heart. Nuclear magnetic resonance imaging was performed using a commercial 1.5 tesla system to define the volume of interest. Phosphorus-31 spectra were obtained from the septum and anteroapical region of the left ventricle in 10 studies. Relative peak heights and areas were determined for high energy phosphates. The mean phosphocreatine to adenosine triphosphate ratio was 1.33 +/- 0.19 by height analysis and 1.23 +/- 0.27 by area analysis. Duplicate measurements in four subjects showed a reproducibility of less than or equal to 10% in three of the subjects. All spectra showed significant signal contribution from the 2,3 diphosphoglycerate in chamber red cells without evidence of skeletal muscle contamination. These results demonstrate the feasibility of image-guided phosphorus-31 spectroscopy for human cardiac studies and indicate the potential of this technique to study metabolic disturbances in human myocardial disease.

Identification of cis and trans-elements involved in the timed control of a Caulobacter flagellar gene.

The genes encoding the structural components of the Caulobacter crescentus flagellum are temporally controlled and their order of expression reflects the sequence of assembly. Transcription of the operon containing the structural gene for the flagellar hook protein occurs at a defined time in the cell cycle, and information necessary for transcription is contained within a region between -81 and -120 base-pairs from the transcription start site. To identify the sequence elements that contribute to the temporal control of hook operon transcription, we constructed deletions and base changes in the 5' region and fused the mutagenized regulatory region to transcription reporter genes. We demonstrate that sequences 3' to the transcription start site do not contribute to temporal control. We confirm that upstream sequences between -81 and -120 base-pairs are necessary for temporal activation, and that transcription also requires sequences at -26 to -46 base-pairs. A specific binding activity for the region between -81 and -122 base-pairs was shown to be temporally controlled, appearing prior to the activation of hook operon transcription. This binding activity was missing from strains containing mutations in flaO and flaW, two genes near the top of the flagellar hierarchy known to be required for hook operon transcription. Thus, the hook operon upstream region contains a sequence element that responds to a temporally controlled trans-acting factor(s), and in concert with a second sequence element causes the timed activation of transcription.

In vivo phosphorus-31 spectroscopic imaging in patients with global myocardial disease.

The goals of this study were to determine whether abnormalities in phosphorus metabolism could be noninvasively detected using phosphorus-31 nuclear magnetic resonance spectroscopy in patients with dilated cardiomyopathy and left ventricular hypertrophy, and whether these patient groups could be distinguished from each other based on parameters obtained using this technique. Seventeen patients and 14 control subjects were studied using nuclear magnetic resonance spectroscopy. Spectra were obtained from the human heart at rest using 3-dimensional spectroscopic imaging as a localization technique. Data were acquired over an average volume of 48 cc in 26.3 minutes using a 2 tesla imaging and spectroscopy unit. The ratio of phosphocreatine to adenosine triphosphate was 0.89 +/- 0.88 (mean +/- standard error) in normal subjects and did not differ significantly in patients with dilated cardiomyopathy or left ventricular hypertrophy. A prominent peak in the phosphodiester region was seen much more frequently in patients with dilated cardiomyopathy, resulting in significantly higher ratios of phosphodiester to phosphocreatine (1.28 +/- 0.35) and phosphodiester to adenosine triphosphate (0.79 +/- 0.18) in this group compared to normal subjects (0.33 +/- 0.08 and 0.29 +/- 0.08, respectively). However, the various patient groups could not be reliably distinguished from each other based on spectral patterns. These studies demonstrate the feasibility of performing phosphorus-31 nuclear magnetic resonance spectroscopic imaging in patients with myocardial disease. The initial results indicate that, under resting conditions, the ratio of phosphocreatine to adenosine triphosphate is not consistently altered in patients with severe global cardiomyopathies or hypertrophy. Phosphodiesters are elevated in some patients with dilated cardiomyopathy, a finding that may signify abnormal phospholipid metabolism in this condition.

Magnetic resonance spectroscopy. Evaluation of ischemic heart disease.

Magnetic resonance spectroscopy (MRS) is a valuable tool for the study of myocardial ischemia. Phosphorus (31P) MRS can detect changes in high-energy phosphates resulting from ischemia and has been used to determine the sensitivity of metabolic changes to ischemia as well as to investigate the metabolic factors important for myocardial dysfunction. The mechanisms mediating postischemic dysfunction have been investigated using 31P MRS, as have interventions to limit metabolic and functional damage from ischemia. These investigations have laid the groundwork for human cardiac studies. While abnormalities following myocardial infarction have been shown in man, further work must be performed to reliably acquire localized spectra under conditions of ischemia.

Study of the syn and anti isomerism of the long-lasting opiate antagonist naloxazone and related compounds.

A 13C-NMR study of the long-acting opiate antagonists naloxazone (I) and naltrexazone (II) and the long-acting opiate agonist oxymorphazone (III) revealed that these compounds are formed as mixtures of their anti and syn isomers. The less crowded anti isomer was found to be the major product in all cases (ca. 80%). N,N-Dimethyl derivatives of naloxazone (IV), naltrexazone (V), and oxymorphazone (VI), which are sterically more crowded than the corresponding unsubstituted hydrazones I-III, were formed as almost exclusively anti isomers. Pure anti isomer of II was obtained as a 1:1 complex with ethanol. No syn-anti equilibration was observed during the 13C-NMR experiment in any of the cases studied. The relevance of our finding about the syn-anti isomerism of the opiate hydrazones to the understanding of their interactions with the opiate receptor is discussed.

Evaluation of suspected musculoskeletal neoplasms using 3D T2-weighted spectral presaturation with inversion recovery.

This study evaluated the use of the Spectral presaturation with inversion recovery (SPIR) technique with T2-weighting in 43 pathologically proven cases of suspected musculoskeletal neoplasm. Both primary and secondary malignant neoplasms as well as benign neoplasms were studied. The MR features exhibited by this technique are discussed. The images were evaluated by two experienced MRI specialists and graded as to utility into one of four categories as compared to conventional T1- and T2-weighted sequences. In the majority of cases this technique was found to be helpful or extremely helpful. The most useful features of this technique were the elimination of chemical shift artifact, the improved ability to evaluate superficial lesions or the extension of lesions into subcutaneous fat, and substantially improved visualization of both bone marrow and bone cortex interfaces. T2-weighted, fat suppressed imaging proved to be a useful new tool for evaluating musculoskeletal neoplasms.

MRI-derived ventricular volume curves for the assessment of left ventricular function.

To assess the utility of double oblique, ECG-gated 1H magnetic resonance (MR) derived volume curves for assessing LV function, cardiac short axis images were acquired with a fast field echo technique. We applied this methodology to assess left ventricular function in three groups: normals, patients with left ventricular hypertrophy, and dilated cardiomyopathy. Six slices with 16-20 phases per RR interval were analyzed, representing the initial 75-80% of the cardiac cycle. For each slice, the endocardial border of the left ventricular (LV) chamber was manually traced. Using Simpson's rule, the total LV volume at a given phase was determined considering the traced area, thickness and position in three-dimensional space of each of the six constituent slices. The calculated volumes were plotted against time and the stroke volume, ejection fraction and cardiac output were determined. The volume vs time plots for the systolic and diastolic portions of the curve were individually fit to third degree polynomials using a least squares approximation. From the fit curves, the following data were extracted: the mean slope (dV/dT) during filling and emptying, and the time to 1/4, 1/3 and 1/2 filling and emptying. These parameters are valuable indices of the functional status of the myocardium; thus, accurate and useful estimates of LV function can be obtained using MRI derived volume curves in normal and abnormal states.

Altered muscle metabolism shown by magnetic resonance spectroscopy in sickle cell disease with leg ulcers.

We performed 31P magnetic resonance spectroscopy of gastrocnemius muscle at rest in 7 normal volunteers and 12 patients with sickle cell disease (7 with leg ulcers and 5 without leg ulcers but with painful crises). We measured intracellular pH and ratios of Pi to ATP, PCr to ATP and PCr to Pi (Pi = inorganic phosphate, ATP = adenosine triphosphate, and PCr = phosphocreatine). Magnetic resonance arteriograms were also performed. Significant differences were found for PCr/Pi ratios between normals and sickle cell disease patients with leg ulcers (p < 0.008). Magnetic resonance arteriograms were normal in volunteers and patients with sickle cell disease. The altered high energy phosphate metabolism in sickle cell disease with leg ulcers is consistent with muscle ischemia or hypoxia.

Method for assessing cardiac function using magnetic resonance imaging.

The authors sought to define a method to use magnetic resonance (MR) to assess cardiac function by obtaining short-axis images of the left ventricle (LV) in humans. Sagittal and axial scout1H MR images were used in the protocol. The long axis of the LV was defined in both planes using the mitral valve and left ventricular apex as references. Based on this double angulation, the acquisition planes were created for a series of parallel short-axis images extending from the base to the apex of the left ventricular cavity. Cardiac images acquired with a fast-field echo technique, six slices with 16-20 phases per RR interval, were analyzed, representing the initial 75-80% of the cardiac cycle. For each slice, the endocardial border of the left ventricular chamber was manually traced. Using Simpson's rule, the total LV volume at a given phase was determined, considering the traced area, thickness, and position in three-dimensional space of each of the six constituent slices. The calculated volumes were plotted against time, and the stroke volume, ejection fraction, and cardiac output were determined. These parameters are clinically significant indices of cardiac function. Accurate and useful estimates of LV function can be obtained using MRI according to this protocol.

Compartmentalized transcription and the establishment of cell type during sporulation in Bacillus subtilis.

An early step in sporulation of the bacterium Bacillus subtilis, is the formation of two compartments in the developing sporangium: the mother cell and the forespore. These compartments differ in their programs of gene expression and developmental fate. The establishment of cell type within this simple developmental program, is accomplished by the compartmentalization of sigma subunits of RNA polymerase. The localization of these sigma factors results in compartment-specific gene expression. Recent experiments have elucidated some of the early steps in the establishment of cell type. After septum formation, the activity of the sigma factor, sigma F, is confined to the forespore compartment. This, in turn, results in the localized expression of another developmental sigma factor, sigma G. The forespore localization of these two sigma factors, establishes the forespore line of gene expression. sigma F and sigma G also regulate mother cell events. sigma F activity in the forespore regulates the proteolytic processing of sigma E within the mother cell compartment. The localization sigma E activity leads to mother cell expression of another sigma factor, pro-sigma K. The proteolytic processing of pro-sigma K to mature sigma K is controlled by the forespore sigma factor, sigma G. Mature sigma K then directs the transcription of mother cell specific genes. Therefore, the initial localization of sigma F activity to the forespore compartment, orchestrates the establishment of cell type in both forespore and mother cell compartments.

Trimethoprim interferes with serum methotrexate assay by the competitive protein binding technique.

Administration of Bactrim (a combination of trimethoprim and sulfamethoxazole) to a patient who also was receiving methotrexate caused a significant increase in apparent plasma methotrexate concentrations as determined by competitive protein binding assay with use of dihydrofolate reductase (EC 1.5.1.3) from Lactobacillus casei as the binding protein. This spurious increase was caused by trimethoprim in the patient's plasma. A plasma trimethoprim concentration of 0.1 mg/L inhibited binding of radiolabeled methotrexate to dihydrofolate reductase by 50%. In contrast, radioimmunoassay for methotrexate was not affected by concomitant administration of trimethoprim. The competitive protein binding assay for methotrexate should not be used in patients being treated with Bactrim or Septra (a similar combination). However, the L. casei competitive protein binding assay technique can be used to assay plasma trimethoprim concentrations with sensitivity to 0.02 mg of trimethoprim per liter.

Effects of K+ on the proton motive force of Bradyrhizobium sp. strain 32H1.

In previous studies, respiring Bradyrhizobium sp. strain 32H1 cells grown under 0.2% O2, conditions that derepress N2 fixation, were found to have a low proton motive force of less than -121 mV, because of a low membrane potential (delta psi). In contrast, cells grown under 21% O2, which do not fix N2, had high proton motive force values of -175 mV or more, which are typical of respiring bacteria, because of high delta psi values. In the present study, we found that a delta psi of 0 mV in respiring cells requires growth in relatively high-[K+] media (8 mM), low O2 tension, and high internal [K+]. When low-[O2], high-[K+]-grown cells were partially depleted of K+, the delta psi was high. When cells were grown under 21% O2 or in media low in K+ (50 microM K+), the delta psi was again high. The transmembrane pH gradient was affected only slightly by varying the growth or assay conditions. In addition, low-[O2], high-[K+]-grown cells had a greater proton permeability than did high-[O2]-grown cells. To explain these findings, we postulate that cells grown under conditions that derepress N2 fixation contain an electrogenic K+/H+ antiporter that is responsible for the dissipation of the delta psi. The consequence of this alteration in K+ cycling is rerouting of proton circuits so that the putative antiporter becomes the major pathway for H+ influx, rather than the H+-ATP synthase.

Positioning of gene products during Caulobacter cell differentiation.

Caulobacter crescentus has one of the simplest known developmental programs that exhibits both temporal and spatial organization. A hallmark of the Caulobacter cell cycle is that the progeny cells that result from each cell division differ from one another with respect to structure and developmental program. The process of establishing asymmetry prior to cell division requires that a number of gene products be targeted to a pole of the predivisional cell and consequently segregated to one of the two progeny. Several products involved in flagellar biogenesis and the chemotaxis machinery are segregated to the swarmer cell. Evidence suggests that the protein product of some fla and che genes is targeted to the incipient swarmer cell pole. In the case of other flagellar genes, it is the mRNA that is apparently segregated to the swarmer cell. Two heat shock proteins, DnaK and Lon are specifically segregated to the progeny stalked cell.